Novel methods and compositions are provided for the biocontrol of plant diseases, in particular diseases causing postharvest decay. The methods and compositions are both protective and curative. Combinations of antagonistic microorganisms and antifungal agents are used.
The U.S. market for biocontrol of tree fruit postharvest diseases could exceed $100 million by the year 2000 (Industrial Bioprocessing, September 1992). In Postharvest News and Information (1991) it was estimated that approximately 25% of harvested fruit and vegetables are lost because of postharvest diseases. Synthetic fingicides have been the primary means for controlling postharvest diseases of fruit and vegetables. However, increased concern of the public over the carcinogenicity of synthetic fungicides, has led to the withdrawal of some fungicides from the market. The development of fungicide-resistance in pathogens has limited chemical fingicides as a means of controlling them.
Control of plant diseases is not a problem confined to the U.S. The European Parliament has voted in favor of a total ban on postharvest treatment of fruit and vegetables with pesticides as soon as this ban becomes feasible. The withdrawal of current fungicides from use in the United States and other parts of the world is creating a large, new market for biological control agents (xe2x80x9cbiocontrolxe2x80x9d). Baker (1987) has defined biological control as xe2x80x9cthe decrease of inoculum or the disease-producing activity of a pathogen accomplished through one or more organisms, including the host plant but excluding man.xe2x80x9d The cost of commercializing a biological control agent is much less expensive than the cost of commercializing a synthetic pesticide because only Tier 1 toxicology tests (Hofstein et al 1994.) are required. Also, if a biological control agent is properly selected, fewer new environmental impact studies are required.
A type of biological control agent is a microorganism that is antagonistic to postharvest pathogens. For example, antagonistic yeasts have been disclosed as effective biocontrol agents for the biological control of postharvest disease (Wilson and El Ghaouth, 1993, 1997). However, microorganisms currently available have not been accepted as providing control comparable to control obtained by the use of synthetic fungicides. Some limitations are due to the microorganisms""inability to cure previously-established infections in the crops and to prevent the resumption of quiescent infections. Improved and broader biocontrol is desirable.
Antifungal hydrolases such as chitinase, xcex2-1,3-glucanase, lysozyme, and lyticase are low molecular proteins that hydrolyze the main components of fungal and yeast cell walls, xcex2-glucan and chitin (Bowles, 1990; Mauch et al., 1988; Schlumbaum et al, 1986; Kendra et al., 1989; Sahai and Manocha, 1993). These enzymes are reported to play a major role in disease resistance of plants against invading pathogens and may be responsible for the biocontrol activity of some microbial antagonists. The action of glucanohydrolases was reported to inhibit fungal growth (Schlumbaum et al., 1986; Sela-Buurlage et al., 1993).
New means of controlling postharvest diseases are needed that are safe, effective, and economically feasible. The present invention provides such means.
The present invention is directed to novel compositions that are combinations of antagonistic microorganisms with antifungal agents, and to methods of preventing or curing plant diseases caused by various postharvest pathogens that cause decay of plants by applying the compositions of the present invention to plants. xe2x80x9cPrevention or curingxe2x80x9d is included under the general term xe2x80x9cbiocontrolxe2x80x9d of plants. Percent of plants infected is a measure of control. Postharvest decay is one of the detrimental phenomena that is controlled by methods and compositions of the present invention.
The combinations of the present invention form a xe2x80x9cbiocontrol cocktail.xe2x80x9d Suitable antagonistic yeasts include those from the following genera: Candida spp; Cryptococcus spp; Pichia spp; Debaryomyces spp; Bulleromyces spp; Sporobolomyces spp; Rhodotorula spp; Aureobasidium spp; Issatchenkia spp; Zygosaccharomyces spp; Dekkera spp; and Hansenula spp. Other suitable microorganisms include bacteria, for example Pseudomonas syringae and Bacillus subtilus. Suitable enzymes and biochemicals include the enzymes chitinase, laminarase, chitosanase, xcex2-1,3-glucanase, lectins; and the biochemicals: xcex2-1,3-glucan, xcex2-1,4-glucan, a polysaccharide of fungal or yeast origin, and polycations such as glycoprotein.
In a preferred embodiment of a composition of the present invention, the antagonistic microorganism is a yeast and the antifungal agent is an enzyme. For example, the combination of the antifungal property of an enzyme, e.g., lysozyme and/or lyticase and the biocontrol activity of an antagonistic yeast, e.g., C. saitoana, wherein the yeast can function against the pathogen in the presence of the enzymes, provides improved consistency and efficacy in controlling postharvest rot (decay). In addition, the combination of antagonistic yeast (C. saitoana) with lysozyme or lyticase offers control of postharvest decay of fruit and vegetables superior to that obtained with an antagonist yeast alone or with an enzyme alone such as lysozyme or lyticase. This improvement and synergism is unexpected.
The combination of antagonistic microorganisms such as a yeast with antifungal enzymes (lysozyme or lyticase) was not expected to successfully work to effect biocontrol of plant pathogens or to produce a synergistic effect because of the known actions of each agent individually. Antifungal hydrolases such as chitinase, xcex2-1,3-glucanase, lysozyme, and lyticase are known to hydrolyze the main components of fungal and yeast cell walls, xcex2-glucan and chitin, and thereby negatively affect the growth of the yeasts such as Candida spp. and of filamentous fungi. Therefore, a combination of an antagonist yeast with antifungal enzymes was expected to impair the biocontrol activity of the selected yeast. However, unexpectedly, the combination produced improved control of pathogens.
Compositions including antagonistic microorganisms such as a yeast (C. saitoana) with antifungal agents such as the enzymes lysozyme or lyticase may be applied to plants either before or after infection because the compositions have both a protective and a curative effect against major postharvest pathogens and consequently offer a level of control of decay better than that of synthetic chemical fingicides.
It is contemplated that a plurality of antagonistic microorganisms may be combined, as long as the microorganisms do not adversely affect the antagonism, viability, or other parameters that are needed for antagonistic microorganism activity according to the present invention. In addition, not only may a plurality of microorganisms be effective in combination with one antifungal agent, but a plurality of antifungal agents may also be effective, provided the plurality of antifungal agents do not deleteriously affect the biocontrol action either of other agents or microorganisms combined with them.
The compositions of the present invention are applied to plants by means such as spraying, drenching, or dipping, which are known in the art. Effective amounts of antifungal agents, e.g. enzymes, have been found to range from 20 xcexcg/ml to about 1000 xcexcg/ml, with about 100 xcexcg/ml being preferred. Effective amounts of yeasts have been found to range from about 106 CFU to about 108 CFU with 108 CFU preferred. It is understood, however, that optimal concentrations will vary with particular situations, and it is well within the level of skill in the art to arrive at optimal formulations by following conventional testing procedures such as those described by the Examples herein.
The complexity of the mode of action displayed by the combined agents of the present invention makes the development of pathogen resistance in the target plants more difficult to achieve and presents a highly complex disease deterrent barrier.
The combination of antagonistic microorganisms with antifungal biochemicals provides effective control against the major rots affecting agricultural commodities such as pome fruits, citrus fruits, vegetables, root crops, stone fruits, tropical fruits, tomato, and other plants.
xe2x80x9cBiocontrol cocktailsxe2x80x9d are compositions of the present invention that are combinations of microorganisms antagonistic to postharvest pathogens that deleteriously affect agricultural commodities, with antifungal agents. The present invention provides a biological protective effect as well as a curative effect.
Fruits and plants that are targets for the methods and compositions of the present invention include pome fruit (e.g., apple, pear); stone fruit (e.g., peach, nectarine, prune); citrus fruit (e.g., orange, lemon, grapefruit, tangerine); root crops (carrots, potato); vegetables (e.g., tomato, bell pepper, cucumber); tropical fruit (e.g., mango, banana, guava, pineapple, avocado); and melon fruit.
An embodiment of the invention is a composition including antagonistic yeasts with antifungal enzymes. Among the enzymes suitable for practice of the invention, hydrolases such as lysozyme and lyticase were found to significantly increase the biological activity of antagonistic yeasts such as C. saitoana that was reported by Wilson and El Ghaouth in U.S. Pat. No. 5,591,429, incorporated herein by reference. Combining yeast with an enzyme exerted an unexpected synergistic biocontrol effect. This combination makes it possible to exploit the biological property of antifungal enzymes and the biocontrol activity of a microbial antagonist. Although antifungal enzymes such as lysozyme and lyticase are inhibitory to postharvest pathogens such as Botrytis cinerea (Table 1), they show no effect on the growth of the yeast C. saitoana (Table 2). Therefore, these ingredients may be combined and the yeast biocontrol activity will be maintained.
Unexpectedly, the combination of antagonistic yeast with lysozyme or lyticase was more effective in controlling infection of apple and citrus than either the antagonist or the enzyme alone (Tables 3 and 4). Increasing the concentration of lysozyme from 100 to 1000 xcexcg/ml resulted in an increase in the effectiveness of the combination (Table 3). In the test conducted with orange fruit, only 30% and 26% of fruit treated with the combination of C. saitoana with lysozyme or lyticase were infected, while 88%, 88%, and 58% of the fruit treated with lysozyme, lyticase, or C. saitoana alone were diseased. All of the control fruit were diseased (Table 4). The same pattern of decay control by the combination was also observed in apple and lemon fruit. (Tables 3 and 4)
In addition to having a protective effect, the compositions of the present invention, e.g. combination of antagonistic yeast with lysozyme or lyticase, also displayed a curative activity against major postharvest pathogens, e.g. apple and citrus fruit (Table 5). The combination of C. saitoana with lysozyme was effective in controlling decay of orange and lemon fruit due to natural infection. The level of disease control obtained with the combination was comparable to that obtained with the recommended fungicide Imazalil (Fungaflor 500EC, 44.6% active ingredient, Janssen Pharmaceutica, Titusville, N.J.). (Table 6).